Mycoplasma Contaminations in the Cell Culture - Background Information, Detection, Treatment

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1 Mycoplasma Contaminations in the Cell Culture - Background Information, Detection, Treatment Dirk Vollenbroich, Ph.D. Minerva Biolabs GmbH

2 Cell Threats Contamination with other cell lines Yeast Fungi Viruses: especially bovine Pestiviruses BVDV Virus of Bovine Virus diarrhea CSFV Virus of the classical swine but also BDV (Borna Disease Virus) Bacteria Mycoplasma

3 Mycoplasma Found in1898 classified as virus Bacteria, origin in gram-positive Bacillus/Lactobacillus/ Streptococcus line, but own class Colloquial: Mycoplasma or PPLO (pleuropneumonia-like organism) Class of Mollicutes (soft skin) with the families: Mycoplasmataceae: Mycoplasma (animal), Ureaplasma (animal) Acholeplasmataceae: Acholeplasma (animal, plant) Spiroplasmataceae: Spiroplasma (plant, anthropodes, rodent) Anaeroplasma (ruminant)

4 Mycoplasma (continued) Pass cellulose- and polyvinyl filter with 0,45 µm pore width Smalles, self-replicating organisms (0,3 to 0,8 µm, 600 kb to 1700 kb (1/5 of E. coli-genome) with approx. 500 genes Need to consume cholesterol, l amino acids, fatty acids, vitamins i and other catabolites Typically arginine, glucose or urea metabolism Lacks cell wall, but has simple plasma membrane extremely flexible in relation to environment, however sensitive in relation to chemical influences resistant to penicillin, contains no peptido p glycan gy wall osmotically unstable Occurring extra cellularly, only in rare cases intracellularly

5 Mycoplasma (continued) Parasites for humans, animals, plants, insects, etc. Effects latent infections of human beings respiratory tract : M. pneumoniae genital tract: M. genitalium, Ureaplasma urealyticum, M. hominis joints: M. fementans, M. arthritidis central nerve system: M. pneumoniae heart: M. pneumoniae Effects on plants: blossom greening (clover, strawberry) yellowing, stall (vine, pear, ster) midget growth (rice) sproud shooting (appel) transfer by cicada and leaf fleas Average cell culture contamination rate: 5 % in industry, 47 % in academics

6 Mycoplasma awaited the development of the cell culture in order to find their actual biological niche.

7 Mycoplasma in the Electron Microscope Bovince cell line MDBK (a) without and (b) with Mycoplasma Source: Lünsdorf & Rohde, GBF Braunschweig BG Chemistry BookletB 004

8 Effects on Cell Culture Inhibition of cell proliferation up to 50% by nutrient withdrawal and secretion of harmful metabolic products McGarrity et al. (1984) In Vitro Cell. Dev. Biol. 20:1 fast glucose reduction and formation of acids => p H shift arginine depletion => inhibition of protein biosynthesis, cell division and growth Influence of immunological reactions (macrophage activation, inhibition of antigen presentation, ti induction of signal transduction) ti Mühlradt et al. (1996) Biochemistry 35:7781 Influence of virus proliferation and the infection rate Nar-Paz et al.(1995) FEMS Microbiol. Lett. 128:63 Cause chromosomal aberrations and multiple translocations McGarrity et al. (1978) In: Mycoplasma infection of cell cultures. Plenum Press S. 213ff Disturbance of the hybridoma technique, contaminated cells become sensitive in HAT medium

9 Effects on Cell Cultures (continued) Accumulation of mycoplasma at cells wall alters cell wall integrity Significant changes in micro array and gene expression profiles bioinformatics.picr.man.ac.uk/experiments/mycoplasma/ Mycoplasma can constitute t up to 50% of the total t protein and 15-30% of the isolated DNA Decrease of the transfection rates by 5% through electroporation Induction of leopard cells (condensation of the chromatins) Influence almost all functions of the host cell metabolism

10 Frequency and Source of Mycoplasma Species Occurring in Cell Cultures (Literature comparison) 20,2 % 25,3 % A. laidlawii others 9% 18% M. argininii 17% M. hyorhinis 20% M. salivarium 5% 36,9 % M. orale 23% M. hominis 5% M. fermentans 3%

11 Sources of Contamination Primary cultures from the original tissue (incidence approximately 4 %) Cross contamination contaminated cultures new cultures from unknown sources, also partly from cell banks virus suspensions, antibody- solutions or other additions of contaminated cell cultures Direct contamination serum (only treated serums, e.g. UV or γ-radiated are presumably mycoplasma free) laboratory instruments, media and reagents, which came into contact with contaminated cultures

12 Sources of Contamination (continued) The User direct entry while handling, usually from the oral flora droplet transfer lacking disinfection careless technical work From:Toni Lindl, Zell-und Gewebekultur

13 Importance of the Mycoplasma Tests Cell cultures offer ideal living conditions to parasitic mycoplasma: contamination is possible at any time Despite titers from 10 7 to 10 8 mycoplasma/ml in cell cultures, no apparent projection referring to the contaminating mycoplasma species and the cell type Microscopically unrecognizable Standard antibiotics can allow contamination levels lower than detection levels, Pen/Strep does not provide protection from contamination!! only each 10th cell culture user regularly tests for mycoplasma contamination!!

14 Instruction for Testing Regulations: FDA Points to Consider (May 1993), Regularien 21CFR USDA federal code #9CFR European Pharmacopoeia 2.6.7, Suppl. 5.8 ICH Guideline for biotechnological/biological products Obliged to test: Master cell cultures, cell cultures, virus stocks, control cell cultures Bioproducts from cell cultures (antibodies, hormons, immune stimulators, blood products from cell cultures) Vaccines for humans and the veterinary field Test necessary for: Editors who are aware of the significance of mycoplasma contamination

15 Fluorescence method Simple, direct indicator for vital mycoplasma Little operative expense, but very time consuming Poor indicator for mycoplasma species with tendencies of extra cellular cell absorption via cytadherent proteins Seminars and experience required Eur. Ph.-listed evidence

16 Culture method Strict requirements for the culture medium and growth conditions (aerobic/anaerobic), generally requires non-standard adjustments for the individual species Extremely long testing times of 1 to 4 weeks Difficult analysis Broth and disk possible Advantage: only living mycoplasma is detected t d Sensitivity: 1 CFU corresponds to average 30 GU M. argininii Source: Mycoplasma Experience Ltd. bar = 1 mm

17 NAT method Nucleic acid amplication test with primers and commercial kits free of choice Eur. Ph , v 5.8, valid since 01. July 2007 Validation must show equality to established methods according sensitivity, specificity, and robustness Can replace indicator methods if sensitivity below 100 cfu/ml Can replace culture method if sensitivity is below 10 CFU/ml Can replace both methods if results are required quickly Cell culture enrichement possible to increase sensitivity

18 Alternative ti Mycoplasma Diagnostic Methods Method Necessary Devices and Evaluation Biochemical Verification Methods combined with luminescence detection Biochemical Verification Methods Adenosinphosphorylase Test (6-MPDR-Test) Enzyme Immuno Verification requires luminescence reader, low sensitivity of approx CFU per test, high demands for sample quality easy usable none / requires indicator cell line, low sensitivity, easily performed ELISA-Reader / specific for mycoplasma species, intermediate sensitivity (10 6 /ml), time intensive

19 Features of Venor GeM Validated according to Eur. Ph , 5.8 Recommended by the WHO More than 100x cited in publications Specific for > 25 mycoplasma species Detection limit 1,5 copies/µl, LOD 95% = 4,5 copies/µl Clear yes/no-result after 3 hours Package sizes: 25, 50, 100 und 250 tests, User friendly aliquotes á25tests tests. Aliquots of Master-Mix can be stored frozen including hot-start Taq possible. Also available for real-time PCR

20 Analysis using Venor GeM 100 bp DNA ladder negative control / internal control amplification copies copies 1000 copies 100 copies 10 copies 1 copy

21 Frequency of Testing New cell and virus cultures Each month for continuous cell lines; each week in cases of laboratory contamination Before each liquid nitrogen storage Upon modification of the cell characteristics In case of problems with result reproducibility

22 Procedure for Contaminated Cell Cultures/ Virus Isolate the culture immediately (incubator and if possible autoclave) Immediately lock and test cryo- conserves Isolate all cryo-conserved samples Inform all possible receipts Standard disinfection of the laboratory Initiate immediately treatment with irreplaceable cells

23 Mycoplasma Elimination Methods Antibiotics average activity: Ciprofloxacin (Ciprobay, Ciproxin), Doxycyclin low activity: Plasmocin, Chloramphenicol, Clindamycin, Azithromycin, Clarithromycin, Tetracyclin, Tiamulin no activity: Penicillin, Streptomycin, Polymyxin, Vancomyin, Erythromycin (only active against some species), Cephalosporine, Sulfametaxol, G418 (Geneticin, Gentamycin-Analogon) Analogon), Bacitracin Complement Fixation Co-Cultivation with Macrophages Physical and chemical methods heat inactivation at C photo inactivation with Hoechst 33258/5-Bromuracil liquid extraction Autoclave

24 Effective Elimination with Mynox Gold Basically no cytotoxicity Highly effective: up to 100% permanent elimination with first treatment Universal for cells Universal for Mycoplasma Low resistance risk Convenient Format Interoperable with other antibiotics

25 Effect of Mynox on Mycoplasma Electron micrographs of mink lung cells (ML cells), contaminated with Mycoplasma hyorhinis Source: M. Özel, Robert-Koch-Institut Berlin

26 Recommendations for Improvement Regularly and sensitive testing (monthly); weekly testing in cases of laboratory contamination Operate free of standards antibiotics Whenever possible: separate the work benches and incubators for handling contaminated and mycoplasma free materials Never use contained cultures; reject immediately or treatedt Disinfect working surfaces and hands with alcoholic spray before and after working procedures, or with the change of the working material Quarantine new cells of any origin and integrate into the laboratory only after testing negative For larger loads of serum, incubate on indicator cells 3T3 or Vero over 4 passages before integration and use, and the test supernatants by means of PCR